Effects of drought stress during critical periods on the photosynthetic characteristics and production performance of Naked oat (Avena nuda L.)

Revealing the effects of drought stress on the photosynthetic characteristics and yield of naked oats (Avena nuda L.) is significant for enhancing the productivity of oats. In this study, a potted experiment consisting of four water levels was conducted in the Bashang area of Hebei Province, China. The drought stress period was established as the continual 8 days during the jointing-heading stage. The aims were to reveal the impacts of drought stress on the photosynthetic characteristics and yield of naked oats during the critical stage. The results showed that the photosynthetic rate (Pn), transpiration rate (Tr), and stomatal conductance (Gs) decreased under all conditions of drought stress. The intercellular CO2 concentration (Ci) decreased under light drought stress, while it increased under moderate and severe drought stress. The initial chlorophyll fluorescence rate (Fo) increased by 9.03–50.92% under drought stress, and the maximum fluorescence rate (Fm) decreased by 8.49–19.73% under drought stress. The photochemical efficiency (Fv/Fm) increased by 10.37–24.12% under drought stress. The yields decreased by 9.5–12.7%, 16.8–27.0% and 44.1–47.7% under light, moderate and severe drought stress during the critical stage, respectively. The grains per panicle decreased by 1.7–12.5%, 8.3–24.3% and 32.7–34.2% under light, moderate and severe drought stress conditions, respectively. The 1000-grain weight decreased by 5.7–8.6%, 12.7–14.5% and 16.8–19.1% under light, moderate and severe drought stress conditions, respectively. The panicle numbers did not vary significantly among the different drought stress treatments. The photosynthetic rate, stomatal conductance and transpiration all had significant positive relationships with the yield of naked oat (P < 0.01). Parameters of PS II except for Fo all had significant positive relationships with the yield of naked oats (P < 0.05). This study is significant for enhancing the production efficiency of naked oat under drought stress.


Materials and methods
Study site, climate and soil data. The experiment was conducted at the Xishungou Station (41° 3′ 54″ N, 114° 4′ 18″ E) of the Zhangjiakou Academy of Agricultural Sciences (Zhangjiakou, China) from 2018 to 2019. The station is characterized by a typical continental climate with abundant solar radiation, warm summers and cold winters. The soil type is chestnut soil, and the detailed soil information is shown in Table 1. The average daily maximum and minimum temperatures were 24.2 °C and 12.8 °C in 2018, respectively, while these values were 23.0 °C and 11.2 °C in 2019, respectively (Fig. 1). Total precipitation during the growing season was 387.6 mm and 238.3 mm in 2018 and 2019, respectively (Fig. 1). The daily maximum temperature during the drought stress treatment period was lower in 2018 with a range of 19.2-26.9 °C, and the range was 21.2-28.1 °C in 2019 (Fig. 1). However, the daily minimum temperature was higher in 2018, with a range of 13.6-19.0 °C, and  Measurements. Photosynthetic rate. The photosynthetic rate of the naked oats leaves was measured at 09:00-11:00 on sunny days using an LC pro + portable full-automatic photosynthetic measurement system (Bio-Scientific, Ltd., Hertfordshire, UK) under natural conditions. The third healthy functional leaf counted from the tip of each staple plant was selected for measurements, and three data points within the same leaf were recorded for each measurement. The parameters obtained were the net photosynthetic rate (P n ), stomatal conductance (G s ), intercellular CO 2 concentration (C i ) and transpiration rate (T r ).  Calculation of tolerance index and mean productivity. To evaluate the impacts of drought stress on naked oat yield, the Tolerance index (TOL) 22 and Mean Productivity (MP) 23 were utilized, and the equations were calculated as follows: where Y c and Y s are the yields under controlled and water stress conditions.
Calculations of the correlations between photosynthetic characteristics and naked oat yield. The correlations between photosynthetic characteristics and naked oat yield were calculated based on Pearson's correlation coefficients: where X i and Y are the photosynthetic indices and naked oat yield. Cov(X, Y) is the covariance between the photosynthetic indices and naked oat yield, and σ Xi and σ Y are the standard deviations of photosynthetic indices and naked oat yield.
Tools for data analysis and plotting. SPSS 10.0 (SPSS, Inc., Chicago, IL, USA) was used for data analysis, and Microsoft Office Excel 2010 (Redmond, WA, USA) was used for plotting.

Results
Effects of drought stress on photosynthetic characteristics. The photosynthetic rates under LS, MS and SS were significantly lower than those of the CK (P < 0.05), and the values decreased by 9.30%, 14.08% and 20.89%, respectively, in 2018, while the decreases in 2019 were 14.43%, 16.85% and 24.55%, respectively ( Fig. 3a,b). The photosynthetic rate was lower in 2019 than in 2018 for each treatment (Fig. 3a,b). With the increase in drought stress, the C i first decreased and then increased (Fig. 3c,d). Compared with the CK, the C i decreased by 12.41% under LS, while it increased by 9.49% under SS, the difference was not significant under MS in 2018 (Fig. 3c). In 2019, the C i under LS increased significantly. The C i under MS and SS was 4.94% and 13.09% higher than that of the CK (P < 0.05), respectively (Fig. 3d). The C i in each treatment was higher in 2019 than in 2018 (Fig. 3c,d). With the increase in drought stress, the T r gradually decreased (Fig. 3e,f). The T r under LS, MS and SS during the 2-year period was significantly lower than that of the CK (P < 0.05). The T r decreased by 10.83%, 41.39% and 45.61% in 2018, respectively (Fig. 3e), and by 21.85%, 29.30% and 45.77% in 2019, respectively (Fig. 3f). The T r was lower in 2019 under all the treatments than in 2018 (Fig. 3e,f). The G s of naked oats decreased as the intensity of drought stress increased (Fig. 3g,h). In 2018, the difference of G s under LS and CK was not significant, while the G s decreased by 35.68% and 78.89% in MS and SS, respectively, compared with that of the CK (Fig. 3g). In 2019, the Gs under LS, MS and SS were 26.04%, 46.88% and 85.42% lower than that of the CK, respectively (P < 0.05) (Fig. 3h). The G s was higher under the CK in 2019 than that in 2018, while the values in other treatments were higher in 2018 (Fig. 3g,h).
Effects of drought stress on photosystem II. The initial fluorescence value (F o ) increased with the aggravation of drought stress (Fig. 4a,b). In 2018, the F o under LS, MS and SS was 9.03%, 9.89% and 14.13% higher than that of the CK, respectively (P < 0.05) (Fig. 4a). In contrast, the F o only increased significantly under MS and SS, and the values were 24.84% and 50.92% higher than those of the CK in 2019, respectively (P < 0.05) (Fig. 4b). The F o was higher in all the treatments in 2019 than in 2018 (Fig. 4a,b). The F m decreased with the increase in drought stress (Fig. 4c,d). The difference of F m under the LS and CK was not significant in 2018, while the F m under MS and SS was 8.49% and 19.73% lower than that of the CK, respectively (P < 0.05) (Fig. 4c). In 2019, the F m under LS, MS and SS were 10.02%, 15.83% and 21.89% lower than that of the CK (P < 0.05) (Fig. 4d). The F m was higher in 2018 under all the treatments than that in 2019 (Fig. 4c,d). The variable fluorescence of PS  (Fig. 4g,h). The F v /F m values under drought stress were lower than that of the CK (Fig. 4i,j). There was no significant difference between the LS and CK (P > 0.05), while the values under MS and SS differed significantly compared with that of the CK (P < 0.05), which decreased by 10.37% and 24.12%, respectively ( Fig. 4i).
In 2019, the F v /F m under LS, MS and SS decreased by 13.3%, 22.44% and 36.83%, respectively (Fig. 4j). The F v /F m was lower in 2019 for each treatment than that in 2018 ( Fig. 4i,j).

Effects of drought stress on the main economic characters.
With the aggravation of drought stress, the plant height of naked oats gradually decreased (Table 2). There was no significant difference between the LS and CK for plant height during the two-year period, while the height decreased by 6.30% and 8.01% in 2018 under MS and SS, respectively (P < 0.05), and the height decreased by 7.88% and 22.16% in 2019, respectively (P < 0.05) ( Table 2). The plant height was almost the same under the CK in two years, while the height was higher under LS and MS in 2018 than in 2019, and it was higher under SS in 2019 (Table 2). Drought stress significantly decreased the panicle length (Table 2). Under LS, the panicle length decreased slightly for the two years, while the length decreased by 16.37% and 12.52% under MS and SS in 2018, respectively (P < 0.05), and decreased by 17.29% and 12.28% in 2019, respectively (P < 0.05) ( Table 2). The panicle length was longer under all the treatments in 2018 compared with that in 2019 ( Table 2). The spikelet number under LS and MS did not   Table 2). The stem number in 2019 was less than that in 2018 under all the treatments ( Table 2).

Effects of drought stress on the yield components.
There was no significant difference in the number of panicles per pot among the drought stress treatments in 2018, while only SS significantly decreased the panicle number by 28.7% compared with the CK in 2019 (Table 3). There were more panicle numbers per pot in 2019 than in 2018 (  Table 3). The yield was higher in 2018 than in 2019 under the CK and SS, and it was higher in 2019 under LS and MS (Table 3). Drought stress significantly decreased the biomass in 2018 (P < 0.05), and the biomass decreased by 6.39%, 18.3% and 21.3% under LS, MS and SS, respectively (Table 3). In 2019, the decrease in amount of biomass was not significant under LS, and the biomass decreased by 14.75% and 17.69% under MS and SS, respectively ( Table 3). The biomass was higher in 2019 than that in 2018 under all the treatments (Table 3). Drought stress also significantly decreased the harvest index (P < 0.05) in both years, and the harvest index was higher in 2018 than that in 2019 under the CK, LS and SS, and it was higher under MS in 2019 (

Relationship between the yield and photosynthetic characteristics of naked oat. The relation-
ship between the yield of naked oat and its photosynthetic characteristics is shown in Fig. 5. The yield of naked oat increased significant with the increase in photosynthetic rate (P < 0.01), and the coefficient of determination (R 2 ) between the rate of photosynthesis and yield of naked oat was 72% under drought stress (Fig. 5a). There was no significant relationship between the yield of naked oat and the intercellular CO 2 concentration (Fig. 5b). Stomatal conductance and transpiration both had significant positive impacts on the yield of naked oat (P < 0.01) (Fig. 5c,d). There was no significant relationship between the yield of naked oat and F o (P > 0.05) (Fig. 5e). The parameters of PS II, including F m (P < 0.05), F v (P < 0.01), F v /F m (P < 0.01) and Fv/Fo (P < 0.05), all have significantly positively relationship with the yield of naked oat (Fig. 5f-h).

Discussion
The response of photosynthetic efficiency to drought stress. Photosynthesis is the basis for crop growth and development, and it is the major factor that determines the composition of crop productivity 24,25 . The decrease in photosynthetic rate under drought stress is a common phenomenon 26 . The G s and P n were found to decrease sharply with the increase in drought stress. The same phenomenon was found in this study because a decrease in the supply of water decreases the G s under drought stress to reduce water loss, and stomatal closure further leads to an insufficient supply of CO 2 , thus, resulting in the reduction of P n 19,27,28 . The trends of change of P n , G s and C i enabled the determination of whether the stomatal factors are restricted 29 . During the early stage of drought stress (or light drought stress), the stomata closed first to reduce water transpiration, thus, preventing CO 2 from entering the leaves 27 . Under moderate and severe drought stress, the concentration of C i gradually increased as the P n and G s decreased, indicating that non-stomatal restriction may gradually became the primary factor of the decrease of photosynthetic rate as the drought stress deepens, which could be owing to the damage of chloroplast structure 30 . However, these conclusions merit further research, which could entail the use of 13 C or 18 O isotope tracers 31 .
The impacts of drought stress on photosystem II. Chlorophyll fluorescence can reflect the primary photosynthetic reaction process, including the absorption of light energy and the transmission of excitation energy and photochemical reaction 32 . The degree of damage caused by stress can be reflected by measuring chlorophyll fluorescence parameters, such as the light utilization in PS II 33 . Under drought stress, plants maintain the balance of water budget by reducing the T r , which is an adaptive way to avoid drought 34 . The F o reflects the degree of damage to the thylakoid membrane with more serious damage of the thylakoid membrane, which induces higher F o values 34 . The F m reflects the electron transfer through PS II. A lower F m reflects a higher degree of thermal damage 34 . F v /F o represents the potential activity of PS II and reflects the activity of PS II center 28 . The F v /F m of plants is typically 0.75-0.85 under non-stress conditions 35,36 , and it will be significantly reduced under conditions of adversity or injury 37 . Our results of the two-year study showed that the F o in oat leaves increased in parallel with the water stress during the critical period because the damage of degree of thylakoid in leaves increased gradually as the water stress deepened. The electrical transmission through PSII in the leaves was inhibited under drought stress conditions, and thus, the F m decreased under water stress. Moreover, water stress reduced the efficiency of capturing light energy in the PSII reaction center of leaves. Thus, the F v /F o and F v /F m in the oat leaves were lower than those in the CK. In addition, more parameters, such as non-photochemical quenching (NPQ) and the fast repetition rate (F m-FRR ), were related to PSII 38,39 . Under drought stress conditions, the intensity of fluorescence decreased, while the relative fluorescence of L-and K-bands in Plectranthus scutel- www.nature.com/scientificreports/ larioides increased 40 . The same phenomenon was also found in other crops 41,42 . The regulation of drought stress for PSII could not be revealed comprehensively with the parameters measured in this study 43 . Nevertheless, this study could explain gaps in the knowledge of the impacts of drought stress on naked oat photosynthesis and yield formation. Different cultivars typically responded differently to drought stress. Previous studies showed that the P n of Bayou3 and Mengyan1 decreased by 42.7% and 34.2% under SS 5,6 , and their level of decline was more dramatic than that of Huazao 2 (used in this study). The Fo increased by 39.0% and 20.3% for Bayou 3 and Mengyan 1, respectively, and the increase was higher than that for Huazao 2 5,6 . The changes of the parameters showed that the cultivar used in this study (Huazao 2) was more drought resistant than Bayou 3 and Mengyan 1.

Effect of drought stress on economic and yield characters of naked oats. Drought stress has been
shown to decrease wheat (Triticum aestivum L.) yield and composition factors 26,44 . In addition, the development of organs is significantly affected under drought stress 45 . In this study, the plant height, panicle length, panicle grain weight, stem number and 1000 grain weight of naked oats all gradually decreased under different amounts of drought stress. The same results were found in previous studies 30,46 . This is because drought stress decreases the accumulation of biomass, and thus, inhibits the growth of oat ears, leaves, stems and roots. However, the accumulation and distribution of dry matter were not determined in this study. Additional research should focus on the accumulation and distribution of dry matter of oat under drought stress conditions. Mild drought had little effect on oat biomass, while moderate and severe drought had a greater effect on oat biomass. Compared with light stress, the yield and yield components of oat decreased more under moderate and severe drought stress because the photosynthetic capacity of the leaves decrease more under moderate and severe drought stress.
The tolerance index (TOL) and mean productivity (MP) values provide information on the stability of yields under different conditions 22 . In this study, we analyzed the TOL and MP under different drought stress conditions in 2018 and 2019. The TOL was higher under LS and MS in 2018 than in 2019. The yield of naked oat was almost the same as that of the CK for both seasons. The higher TOL under 2018 was owing to the distribution of precipitation under LS and MS during the oat growing period. The TOL under SS was almost the same for both seasons, implying that the SS caused irreversible damage to the naked oat. The MP was higher in 2018 than in 2019 under LS and SS, and there was more precipitation during the growing season in 2018. This implies that the cultivar used was not very resistant to drought.
We further analyzed the relationships between the yield of naked oat and the photosynthetic characteristics, which showed that the photosynthetic rate was significantly related to oat yield (P < 0.01). Typically, P n is an instantaneous value, and a higher P n during the crop growth period might not produce higher yields 47 . When the crops were subjected to stress, the activity of P n was lower during the entire growing period, and the yield decreased significantly 24 . The parameters related to PS II also significantly positively correlated with the yield of naked oat. In all, the drought stress decreases the efficiency of photosynthesis and the components of naked oat yield.
Limitations of the study. There are some limitations in this study. First, the research was conducted on only one cultivar, and the amount of drought resistance varies for different cultivars. Therefore, more cultivars should be considered in future research. Secondly, previous studies showed that the indices, such as the activities of superoxide dismutase (SOD) and peroxidase (POD) and the content of malondialdehyde (MDA), would also change under drought stress conditions 48 . However, these parameters were not measured in this study. Future research should include more parameters. Owing to the limited conditions, the experiment only controlled the soil water content and established the degree of soil drought stress based on it. However, the actual degree of stress of the oat plants was not measured. In addition, only photosynthesis, fluorescence and laboratory tests were performed in this experiment, and changes in the physiological indices related to drought stress of oat were not measured. The next step is to determine the degree of stress of oat based on the leaf water potential and to determine the indicators related to drought stress related, such as the activities of superoxide dismutase (SOD) and peroxidase (POD) and the content of malondialdehyde (MDA) among others.

Conclusions
Drought stress decreased the P n , T r , G s under all the drought stress conditions during the critical period in which water was required. The C i decreased under light LS, while it increased under MS and SS. The F o and F v /F m increased under drought stress, while the F m decreased under drought stress. The oat yield decreased by 9.5-47.7% under drought stress conditions. Grains per panicle decreased 1.7-34.2% under drought stress conditions. The 1000-grain weight decreased by 5.7-19.1% under drought stress conditions. Drought stress had no significant impacts on the panicle numbers per pot. The P n , T r , and G s all positively correlated with the yield of naked oat. The parameters of PSII, except for F o , all significantly positively correlated with the yield of naked oat (P < 0.05). This study provides additional knowledge on the different levels of drought tress on the production of naked oats in the Bashang area. www.nature.com/scientificreports/